Project Summary/Abstract Lysophospholipases A1 and A2 (LYPLA1 and LYPLA2) are serine hydrolases responsible for the hydrolysis of lipid mediators derived from glycerolipids (1-3). These enzymes were first identified as lysophospholipid (LPL) hydrolases, with distinct substrate specificities despite 81% sequence similarity (68% identity). LPLs elicit a myriad of physiological effects. As a result, excessive levels of LPLs have been implicated in numerous pathologies, including atherosclerosis, hyperlipidemia, metabolic syndrome, and cancer (12-18), suggesting lysophospholipase activity is crucial for the prevention of these diseases. Endocannabinoids and their prostanoid metabolites, prostaglandin glyceryl esters (PGGs), are also derived from glycerolipids and play a role in a variety of signaling mechanisms, namely inflammatory responses, vasoregulation, muscle contraction, and nociception (19-22). We have identified LYPLA2 as the major PGG hydrolase in human cancer cells, whereas LYPLA1 completely lacks this activity. This suggests further divergences between these enzymes, as well as a role for LYPLA2 in endocannabinoid metabolism (2). Additionally, the lysophospholipases have recently been identified as acyl protein thioesterases (3, 23), able to hydrolyze conjugates of lipids to proteins. Lipids, namely palmitic acid, are known to conjugate to a plethora of proteins to regulate their function, binding interactions, and subcellular location (24-26). Despite the significance of protein palmitoylation in each of these processes, the only enzymes responsible for the dynamic regulation of this post-translational modification, LYPLA1 and LYPLA2, are poorly understood. Although the lysophospholipases have been implicated in the hydrolytic metabolism of each of these substrate classes, the full extent of the lipid and lipid-modified protein substrate pools as well as the molecular basis for substrate selectivity of these enzymes remains unclear. I hypothesize that that unique structural features of LYPLA2 dictate and modulate its distinct substrate selectivity in vitro and in intact cells. To address this hypothesis, I will use recombinant protein to investigate key structural motifs important for substrate binding. We have solved the crystal structure of LYPLA2, and are using its provided structural information as a basis for the identification of important substrate-binding determinants. Results from these experiments will be used to identify uncharacterized LPL and palmitoylated proteins substrates of the lysophospholipases. I will use three general approaches for these experiments: 1) Characterize the molecular determinants of substrate binding in the LYPLA2 active site; 2) Evaluate the role of lysophospholipases in modulating cellular levels of bioactive lipids using a CRISPR-Cas9 knockout cell line; and 3) Identify protein substrates of LYPLA1 and LYPLA2 depalmitoylation. This research proposal will assess the structural mechanism of LYPLA2 and substrate interactions, as well as identify biologically relevant lipid or lipid-modified substrates of this enzyme to elucidate its role in the regulation of lipid homeostasis.